The objective of this project is the determination of the biochemical basis for the apparent "aging" of a protein, bacterial luciferase, which seems to result from enzymatic turnover in vivo. The synthesis of luciferase is rapidly "autoinduced" at a specific time during late log phase growth in luminous bacterial cultures, so that luciferase represents ca. 5% of the soluble protein in the cells as they enter stationary phase. In wild-type cultures, the luminescence in vivo and the extractable luciferase activity assayed in vitro then fall sharply during stationary phase (t1/2 approximately 30-60 min). Certain mutants appear to lack one of the substrates for the light-producing reaction catalyzed by luciferase, a long-chain saturated aldehyde, and thus the enzyme does not turn over by the luminescent pathway in vivo; the extractable luciferase from these aldehyde-deficient "dark" mutants has approximately twice the specific activity of the enzyme from wild-type cells, and furthermore appears to be stable in vivo for many hours during stationary phase. The lower specific activity and decreased in vivo stability of the wild-type enzyme are apparently the result of some post-synthetic modification which is related to turnover of the enzyme in the luminescent reaction. Our overall objectives include the determination of the cause of the modification, the chemical basis for the decreased specific activity, and the relationship of the modification to the increased susceptibility to the cells' degradative system. The specific objective of the 1980-81 year is to apply the pulse-chase and antibody techniques we have developed to an in-depth analysis of the specific rates of synthesis and degradation of luciferase in cells of the wild type, aldehyde deficient mutants, and mutants having lesions in the luciferase subunit structural genes.